The broad, long-term goal of this project is to demonstrate the feasibility of using rat embryonic stem cells (ESCs) to create targeted knock-out rat models. The lack of rat ESCs has been a major impediment to genetically manipulating the rat genome to create specific rat models and the recent availability of this biological resource opens the way to increasing the rat's utility as an animal model system. The specific objective of this application is to demonstrate proof-of-concept by creating a Pkd1 conditional knock-out rat. The central hypothesis is that rat ES cells will be amenable to standard knock-out technology that has been developed successfully in the mouse and that the ability to target and specifically knock-out genes known to cause human disease will allow appropriate rat models to be developed. In addition, these rat models will provide improved tools for studying disease and for testing therapeutics. The rationale for creating a Pkd1 rat knock-out is that polycystic kidney disease (PKD) is a major human health issue, no Pkd1 rat models currently exist and most available rodent models of PKD do not entirely mimic human autosomal dominant PKD providing less than optimal systems for translational studies aimed at evaluating treatment options. Successful creation of a Pkd1 knock-out rat will set the stage for standardizing the methodology for knocking out any disease gene of interest in the rat. Three Aims are proposed: 1) produce a targeted conditional gene knock-out allele of the rat Pkd1 gene in rat ES cells, 2) create a transgenic rat line carrying a transgene for Cre recombinase driven by human PKD1 regulatory elements under inducible control of tamoxifen, and 3) use these lines to create new rat models for PKD and critically characterize them to evaluate how faithfully they mimic human autosomal dominant PKD. Successful completion of the proposed studies will demonstrate the feasibility of using rat ES cells to knock-out genes of interest in the rat to create relevant models of human disease. The ability to manipulate the rat genome in a targeted manner will have a profound impact on the utility of rats as animal models for biomedical research, with widespread applications for both basic research studies and pre-clinical translational studies to evaluate treatments and therapeutics. PUBLIC HEALTH RELEVANCE: Animal models that mimic human disease are important tools for studying disease processes and evaluating therapeutic treatments and rats are particularly good model systems due to their large size (compared to mice) and physiological similarities to humans. In this proposal, newly available biological reagents (rat embryonic stem cells) will be used to create a new rat model for polycystic kidney disease (PKD), a prevalent genetically- based disorder estimated to affect an estimated 12 million individuals worldwide. The proposed experiments will not only result in improved animal models to study PKD but they will demonstrate the feasibility of genetically manipulating the rat genome to be able to target any gene of interest in order to create new rat models to study human disease.